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Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction.

Wang SJ, Tsai A, Wang M, Yoo S, Kim HY, Yoo B, Chui V, Kisiel M, Stewart B, Parkhouse W, Harden N, Krieger C - Biol Open (2014)

Bottom Line: We provide evidence that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle.We also show that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain.These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada.

No MeSH data available.


Transgenic Hts localizes in muscle nuclei.(A–C′″) High magnification views of nuclei (single sections taken within the nucleus) in muscles 6 or 7 in abdominal segment 4, stained with 1B1 (green), anti-phospho-adducin (red) and the DAPI nuclear marker (blue). Bottom panels show cross-sectional views. (A–A′″) When over-expressing wild-type Hts in the muscle with mef2-Gal4, Hts immunoreactivity is observed in the nucleus as discreet puncta, with accumulations in the nucleolus sometimes observed. However, phosphorylated Hts, detected with an antibody against adducin phosphorylation at the conserved site in the MARCKS-homology domain, is not observed. Note that the immunoreactivities of Hts and phospho-adducin overlap outside of the nucleus. (B–B′″) Non-phosphorylatable Hts shows a similar nuclear distribution pattern as wild-type Hts. (C–C′″) Surprisingly, phospho-mimetic Hts is also observed in the nucleus, though it forms abberant accumulations that differ from the discreet puncta seen with wild-type and non-phosphorylatable Hts. Note that unlike with wild-type Hts expression, elevations in phospho-adducin levels in the muscle are not detected by the phospho-adducin antibody when expressing the non-phosphorylatable and phospho-mimetic hts transgenes as they contain alterations of the target serine residue. Scale bar in Panel C′″ represents 10 µm (for A–C′″).
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f05: Transgenic Hts localizes in muscle nuclei.(A–C′″) High magnification views of nuclei (single sections taken within the nucleus) in muscles 6 or 7 in abdominal segment 4, stained with 1B1 (green), anti-phospho-adducin (red) and the DAPI nuclear marker (blue). Bottom panels show cross-sectional views. (A–A′″) When over-expressing wild-type Hts in the muscle with mef2-Gal4, Hts immunoreactivity is observed in the nucleus as discreet puncta, with accumulations in the nucleolus sometimes observed. However, phosphorylated Hts, detected with an antibody against adducin phosphorylation at the conserved site in the MARCKS-homology domain, is not observed. Note that the immunoreactivities of Hts and phospho-adducin overlap outside of the nucleus. (B–B′″) Non-phosphorylatable Hts shows a similar nuclear distribution pattern as wild-type Hts. (C–C′″) Surprisingly, phospho-mimetic Hts is also observed in the nucleus, though it forms abberant accumulations that differ from the discreet puncta seen with wild-type and non-phosphorylatable Hts. Note that unlike with wild-type Hts expression, elevations in phospho-adducin levels in the muscle are not detected by the phospho-adducin antibody when expressing the non-phosphorylatable and phospho-mimetic hts transgenes as they contain alterations of the target serine residue. Scale bar in Panel C′″ represents 10 µm (for A–C′″).

Mentions: Prior studies have shown that Dlg localization at the postsynaptic membrane of larval NMJs is disrupted through phosphorylation by either PAR-1 or CaMKII (Koh et al., 1999; Zhang et al., 2007). We previously provided evidence that postsynaptic over-expression of Hts, using GS13858, results in elevated PAR-1 and CaMKII protein levels throughout the muscle, thereby promoting Dlg phosphorylation and delocalization (Wang et al., 2011). Interestingly, mammalian α-adducin can shuttle between cell-cell junctions and the nucleus in epithelia due to the presence of a bipartite nuclear localization signal (NLS) in the MARCKS-homology domain and a nuclear export signal (NES) in the neck region (Chen et al., 2011). In the nucleus, α-adducin is required for proper spindle assembly and mitotic progression (Chan et al., 2014; Chen et al., 2011). Upon determining that both the NLS and NES sequences are conserved in Hts, we wondered if the Drosophila adducins are present in muscle nuclei where they can affect other processes such as gene expression. In wild-type body wall muscles, Hts nuclear immunoreactivity was not readily detectable (data not shown). However, when wild-type Hts was over-expressed in the muscle, Hts immunoreactivity was observed in nuclei as discreet puncta (Fig. 5A′).


Phospho-regulated Drosophila adducin is a determinant of synaptic plasticity in a complex with Dlg and PIP2 at the larval neuromuscular junction.

Wang SJ, Tsai A, Wang M, Yoo S, Kim HY, Yoo B, Chui V, Kisiel M, Stewart B, Parkhouse W, Harden N, Krieger C - Biol Open (2014)

Transgenic Hts localizes in muscle nuclei.(A–C′″) High magnification views of nuclei (single sections taken within the nucleus) in muscles 6 or 7 in abdominal segment 4, stained with 1B1 (green), anti-phospho-adducin (red) and the DAPI nuclear marker (blue). Bottom panels show cross-sectional views. (A–A′″) When over-expressing wild-type Hts in the muscle with mef2-Gal4, Hts immunoreactivity is observed in the nucleus as discreet puncta, with accumulations in the nucleolus sometimes observed. However, phosphorylated Hts, detected with an antibody against adducin phosphorylation at the conserved site in the MARCKS-homology domain, is not observed. Note that the immunoreactivities of Hts and phospho-adducin overlap outside of the nucleus. (B–B′″) Non-phosphorylatable Hts shows a similar nuclear distribution pattern as wild-type Hts. (C–C′″) Surprisingly, phospho-mimetic Hts is also observed in the nucleus, though it forms abberant accumulations that differ from the discreet puncta seen with wild-type and non-phosphorylatable Hts. Note that unlike with wild-type Hts expression, elevations in phospho-adducin levels in the muscle are not detected by the phospho-adducin antibody when expressing the non-phosphorylatable and phospho-mimetic hts transgenes as they contain alterations of the target serine residue. Scale bar in Panel C′″ represents 10 µm (for A–C′″).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4265757&req=5

f05: Transgenic Hts localizes in muscle nuclei.(A–C′″) High magnification views of nuclei (single sections taken within the nucleus) in muscles 6 or 7 in abdominal segment 4, stained with 1B1 (green), anti-phospho-adducin (red) and the DAPI nuclear marker (blue). Bottom panels show cross-sectional views. (A–A′″) When over-expressing wild-type Hts in the muscle with mef2-Gal4, Hts immunoreactivity is observed in the nucleus as discreet puncta, with accumulations in the nucleolus sometimes observed. However, phosphorylated Hts, detected with an antibody against adducin phosphorylation at the conserved site in the MARCKS-homology domain, is not observed. Note that the immunoreactivities of Hts and phospho-adducin overlap outside of the nucleus. (B–B′″) Non-phosphorylatable Hts shows a similar nuclear distribution pattern as wild-type Hts. (C–C′″) Surprisingly, phospho-mimetic Hts is also observed in the nucleus, though it forms abberant accumulations that differ from the discreet puncta seen with wild-type and non-phosphorylatable Hts. Note that unlike with wild-type Hts expression, elevations in phospho-adducin levels in the muscle are not detected by the phospho-adducin antibody when expressing the non-phosphorylatable and phospho-mimetic hts transgenes as they contain alterations of the target serine residue. Scale bar in Panel C′″ represents 10 µm (for A–C′″).
Mentions: Prior studies have shown that Dlg localization at the postsynaptic membrane of larval NMJs is disrupted through phosphorylation by either PAR-1 or CaMKII (Koh et al., 1999; Zhang et al., 2007). We previously provided evidence that postsynaptic over-expression of Hts, using GS13858, results in elevated PAR-1 and CaMKII protein levels throughout the muscle, thereby promoting Dlg phosphorylation and delocalization (Wang et al., 2011). Interestingly, mammalian α-adducin can shuttle between cell-cell junctions and the nucleus in epithelia due to the presence of a bipartite nuclear localization signal (NLS) in the MARCKS-homology domain and a nuclear export signal (NES) in the neck region (Chen et al., 2011). In the nucleus, α-adducin is required for proper spindle assembly and mitotic progression (Chan et al., 2014; Chen et al., 2011). Upon determining that both the NLS and NES sequences are conserved in Hts, we wondered if the Drosophila adducins are present in muscle nuclei where they can affect other processes such as gene expression. In wild-type body wall muscles, Hts nuclear immunoreactivity was not readily detectable (data not shown). However, when wild-type Hts was over-expressed in the muscle, Hts immunoreactivity was observed in nuclei as discreet puncta (Fig. 5A′).

Bottom Line: We provide evidence that Hts promotes the phosphorylation and delocalization of Dlg at the NMJ through regulation of the transcript distribution of the PAR-1 and CaMKII kinases in the muscle.We also show that Hts interactions with Dlg and PIP2 are impeded through phosphorylation of the MARCKS-homology domain.These results are further evidence that Hts is a signaling-responsive regulator of synaptic plasticity in Drosophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC V5A 1S6, Canada.

No MeSH data available.